1. Field of the Invention
This invention relates to a method for inactivating parasites in blood and blood products by incubating a mixture of the blood or blood product, a phthalocyanine dye and a quencher and optionally irradiating this mixture with red light. An advantage of the inventive method is that the treatment, when red light is applied, leads to the inactivation of lipid enveloped virus, also contained in the blood or blood product. A further advantage of the instant method is that while the parasites and lipid enveloped viruses are inactivated, blood cells and labile blood proteins contained in the blood or blood product, are not adversely affected.
2. Description of Related Art
It is estimated that 16 to 18 million people are infected world-wide with Chagas disease. This disease is caused by the parasite Trypanosoma cruzi and is endemic to Latin America. A large proportion of immigrants from Latin America to the United States are from areas where the prevalence of the pathogenic agent T. cruzi is high. It is estimated that at least 50,000 people infected with T. cruzi have emigrated to the U.S.A. In addition to insect vectors, another route of infection is transfusion and this is an emerging problem in the United States. Often the infection caused by T. cruzi is chronic and the majority of carriers initially display only mild symptoms. The infectious form of the parasite, trypomastigotes, circulate in the infected individual's blood and is capable of surviving the blood banking process and storage. No serological test for T. cruzi in blood banks has been approved by the FDA for use in the U.S.A. at this time. The inadequacy of identification and screening methods makes it imperative to inactivate the parasite in transfused blood.
While the most serious form of malaria, caused by Plasmodium falciparum, is usually transmitted by a mosquito vector, it may also be transmitted by blood transfusion from asymptomatic donors. Almost all blood components, including red cells, platelet concentrates, white cells, cryoprecipitates and fresh plasma transmit malaria. Malaria parasites can survive storage in red blood cells at 2 degrees to 6 degrees for days to weeks or even years. The FDA's Blood Products Advisory Committee has issued recommendations for deferring blood donors at increased risk for malaria, however, these recommendations apply only to donations containing intact red blood cells. Donations used for preparing plasma, plasma components, or derivatives devoid of intact red blood cells are excluded from these regulations. Consequently, absolute safety from transfusion derived malaria is not insured. It is expected that increased immigration and travel from malaria endemic areas will intensify the risk of malaria through transfusion of red blood cell concentrates (RBCC) and platelet concentrates (PC).
Transmission of pathogenic viruses by blood transfusion has been reduced in recent years by serological screening for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV). However, absolute safety has not been achieved and the risk of HBV, HCV and HIV-1 transmission in the USA with a single blood unit has been estimated at 0.0005%, 0.03% and 0.0005%, respectively (R. Y. Dodd, "The Risk of transfusion-transmitted infection", N. Eng. J. Med., 327, 419-21, 1992). Patients who received a large number of RBCC are at a much higher risk of virus transmission. Other viruses of concern in patients with compromised immune systems are cytomegalovirus (CMV) and parvovirus.
Sterilization appears to be the best way to ensure a very high level of safety in transfusion of blood and its components. Currently, all blood products are available in sterilized forms with the exception of red blood cell and platelet concentrates. Sterilization of cellular blood components presents a unique challenge because cell structure and function are disrupted more easily than those of individual proteins. Various approaches have been taken for virus sterilization of red blood cells (RBC) and platelets (B. Horowitz and J. Valinsky, "Inactivation of viruses found with cellular blood components", Biotechnology of Blood, J. Goldstein (ed.), pp. 431-52, Buttworth-Heinemann, Stoneham, 1991). However, favorable results were obtained only with photodynamic treatment (PDT) (J. L. Matthews et al., "Photodynamic therapy of viral contaminants with the potential for blood banking applications", Transfusion 28, 81-83, 1988). As a result, almost all the efforts are now focused on this approach.
One approach which has been used to sterilize blood and its components is to use psoralens which target nucleic acids and are activated by UVA light. Unfortunately, this approach cannot be used to sterilize RBC. UVA is not effective because of the strong absorption by hemoglobin.
A second approach involves the use of phthalocyanines, which are activated by light in the red light region (650-700 nm). This approach is essentially as set forth in U.S. Pat. Nos. 5,120,649 and 5,232,844 and copending application Ser. Nos. 08/031,787, 08/364,031 and 08/344,919, the entire disclosures of which patents and applications are hereby incorporated by reference. Activation of phthalocyanines by red light in the presence of oxygen is known to result in the disruption of viral membranes. However, nothing is known about the ability of these compounds to inactivate blood borne parasites, particularly protozoa.
Currently, genetian violet (GV) is the only effective agent which may be used for the chemoprophylaxis of T. cruzi in endemic areas. This phenylmethane dye is composed of 96% hexamethylparasaniline (crystal violet). It is used at a final concentration of 0.6 mM. The dye is reduced in the organism, forming a carbon centered free radical, which is able to remove oxygen from other molecules or to be added across unsaturated bonds. The carbon-centered free radical can also autooxidize, producing a superoxide anion radical (O.sub.2). The latter is converted into H.sub.2 O.sub.2 by superoxide dismutase. T. cruzi is sensitive to H.sub.2 O.sub.2 since the parasite is deficient in catalase and reduced glutathione (GSH), which degrade peroxide. The interaction between O.sub.2 and H.sub.2 O.sub.2 generates OH, a highly toxic radical. The presence of light enhances this reaction several fold and reducing agents such as ascorbate increase H.sub.2 O.sub.2 generation. A combination of light exposure and ascorbate will kill parasites using a lesser amount of genetian violet (0.4 mM). The absorption maximum for GV is above 400 nm (R.D. et al., "Light-enhanced free radical formation and trypanocidal action of gentian violet (crystal violet)", Science, 229, 1292-95, 1983). GV has proven to be effective in the inactivation of all parasite stages (amastigotes, trypomastigotes and epimastigotes). RBC survival using .sup.51 Cr as well as blood biochemistry upon storage with GV did not show deleterious effects.
However, there are several side effects associated with GV. First, GV is known to cause microagglutination of the red blood cells. Microagglutination is the clumping of the red blood cells. In vitro, this effect is caused by immunoglobulins (IgG) binding to the red blood cell and the effect is observed when viewing a sample of blood under a microscope. A second side effect which is observed when GV is used as chemotherapeutic agent is rouleaux. Rouleaux describes a condition wherein the red blood cells are aligned on top of each other analogous to a stack of coins. It is observed when the blood is collected and allowed to stand in a tube or in a thick portion of blood.
Finally, there are also morphological changes in platelet mitochondria and hemostatic impairment. In recipients, GV turns the blood into a purple color which may stain the skin and mucosa. Furthermore, there is a carcinogenic effect in rodents. Controlled studies to better understand possible toxic effects of this drug are not currently available.
Additionally, antifungal agents with trypanocidal activity have been used to inactivate T. cruzi in blood. These include amphotericin B, imidazole derivatives, .beta.-lapachone and 2-nitrodesmethyl imipramine. No human studies have been reported so far for any of these drugs.
There are currently no methods known in the art to inactivate P. falciparum in RBCC. Merocyanine 540 has been reported to reduce the concentration of parasitized RBC by 3 log.sub.10 when exposed to light (D. M. Smith et al., "Evaluation of merocyanine 540-sensitized photoirradiation as a method for purging malarially infected red cells from blood", J. Infect Dis., 163 1312-17, 1991). However, because there is significant overlap between the absorption spectrum of merocyanine 540 and that of hemoglobin, this dye is not suitable for use in RBCC.
Phthalocyanines (Pc) are porphyrin-like synthetic pigments with a macrocycle made up of four isonindole units linked by nitrogen atoms. ##STR1## For metals at an oxidation state higher than 2 there are axial ligands which can vary. R can be any substituent, usually sulfonic acid residues.
Phthalocyanines are analogues of porphyrins, with aza nitrogens replacing the methylene bridges and with benzene rings fused onto the pyrrole units. Pc can be derivatized in three ways: substitutions on the benzene rings; changing the central metal ligand; and axial ligands bound to the metal when its valency exceeds 2. Pc are particularly suited for work in RBCC because their high extinction coefficient (&gt;10.sup.5 /mole-cm) at about 680 nm is far removed from that of hemoglobin. When substituted with diamagnetic metals, Pc have long lived exited triplet state and can generate singlet oxygen at a high quantum yield (I. Rosenthal et al., "The role of molecular oxygen in the photodynamic effect of phthalocyanines", Radiat. Res. 107, 136-42, 1968). For a recent review of the photochemistry of phthalocyanines see the article by E. Ben-Hur, "Basic photobiology and mechanisms of action of phthalocyanines", Photodynamic Therapy: Basic Principles and Clinical Applications, B. W. Henderson and T. J. Dougherty (eds.), 63-67, Marcel Dekker, New York, 1992).
In view of the foregoing, it should be clear that there remains a definite need in the art for a method capable of inactivating blood borne parasites without damaging blood cells or labile blood proteins or causing other effects that would be deleterious for the donor or recipient patient.